Deconstructing and recapitulating complex biological processes requires multidimensional control of molecular function. Optogenetics has emerged as a versatile means of achieving this capability, as demonstrated by the impact of channelrhodopsins and halorhodopsins on neuroscience. Developmental and regenerative biology would also be transformed by new optogenetic technologies, as tissue formation requires the coordination of multiple signaling pathways in space and time. To date, nearly all non-rhodopsin optogenetic systems have relied on proximity-based mechanisms to control protein function, using natural photoreceptors such as phytochromes, cryptochromes, and light- oxygen-voltage (LOV) domains. While this methodology has yielded valuable tools, is it not broadly applicable across the proteome. Allosteric optical control is another powerful means of regulating protein activity in space and time. However, relatively few examples of such optogenetic systems have been reported to date, likely due to the challenge of developing functional photoreceptor-signaling protein chimeras. Our project seeks to address this challenge by recapitulating how photoreceptors likely evolved in nature: the random insertion of light-sensing domains into signaling proteins and the selection of photoresponsive variants. In particular, we envision that transposon technologies could greatly expedite optogenetic engineering by bypassing the bottleneck of evaluating individual photoreceptor- functionalized constructs. We will employ Tn5 and Tn7 transposase-mediated insertional mutagenesis to create large random libraries of photoreceptor-signal protein chimeras, and we will identify photoresponsive variants using cell- based reporters and flow cytometry (Aim 1). We will then apply directed evolution and targeted mutagenesis to optimize these optogenetic tools and evaluate their efficacy in zebrafish models (Aim 2). Our proof-of-concept studies focus on LOV domains, taking advantage of their compact size, structural and functional diversity, and amenability to protein engineering. We will apply these microbial and plant-derived photoreceptors to Smoothened (SMO) and GLl1, canonical regulators of the Hedgehog signaling pathway, and we evaluate photoresponsive LOV-SMO and LOV-GLI1 constructs in zebrafish embryos. We anticipate that our transposon-based strategy will be broadly applicable to functionally diverse proteins, accelerating the development of new optogenetic tools and expanding the scope of optobiology. Our long-term goal is to assemble an optogenetic toolbox for manipulating the molecular pathways that regulate tissue patterning and regeneration (e.g., Wnt, BMP, and Notch signaling), optically intercepting multiple points within each pathway.